Sex hormone-binding globulin (SHBG), the specific plasma carrier for sex steroids, regulates hormone bioavailable fraction and estrogen signalling system in breast cancer cells. A common single nucleotide polymorphism in the human SHBG gene results in an amino acid substitution (Asp327Asn), which introduces an additional N-glycosylation site that has been associated with reduced breast cancer risk in postmenopausal women. Since wild-type SHBG was demonstrated to interfere with estradiol signalling, and to inhibit estradiol-mediated proliferation and anti-apoptosis, the present study compares the ability of wild-type and D327N SHBG in modulating estradiol effects in MCF-7 breast cancer cells. Recombinant wild-type and D327N SHBGs were obtained from CHO cells transfected with the two different cDNAs. Recombinant proteins were evaluated as far as their ability to bind estradiol and to interact with cells was concerned; while no difference in estradiol binding was observed, the D327N SHBG bound to MCF-7 cells at a significantly greater extent than wild-type protein. Both proteins were than investigated for their capacity to induce the second messenger cAMP in MCF-7 cell; the levels of cAMP reached after D327N SHBG treatment was again significantly higher that that reached after wild-type SHBG treatment. As well, D327N SHBG was significantly more effective in inhibiting estradiol-induced phosphorylation of Erk 1/2 than wild-type protein. Last, MCF-7 cell proliferation was studied; both SHBGs inhibited estradiol-induced proliferation, but again the D327N form resulted more effective. In conclusion, the polymorphism Asp327Asn of human SHBG confers to this form of protein a protective action in breast cancer, and here for the first time we delineate the mechanism of action, demonstrating that D327N SHBG is more effective than the wild-type protein in modulating estradiol signalling to breast cancer cells.